Gene therapy entails the use of genetic information as the pharmacologic agent. While originally conceived as a means of treating hereditary disease, gene therapy is now recognized as a powerful tool for delivering therapeutic mRNA or proteins for local and/or systemic use (see, e.g., Friedmann, Science, 244, 1275-1281 (1989); Miller, Nature, 357, 455-460 (1992)). Generally, there are two approaches to gene therapy: ex vivo and in vivo. In the ex vivo approach, cells removed from a host are genetically modified in vitro before being returned to the host (see, e.g., U.S. Pat. No. 5,399,346 (Anderson et al.)). In the in vivo approach, the genetic information itself is transferred directly to the host, without employing any cells as a vehicle for transfer.
Both approaches have been employed to transfer a so-called "therapeutic" gene to a host. Broadly considered, a therapeutic gene is a gene that corrects or compensates for an underlying protein deficit or, alternately, a gene that is capable of regulating another gene, or counteracting the negative effects of its encoded product, in a particular disease state, condition, disorder or syndrome. For instance, the ex vivo approach has been used for the modification of T lymphocytes in the treatment of adenosine deaminase deficiency, modification of hepatocytes in the treatment of familial hypercholesterolemia, and modification of tumor-infiltrating lymphocytes in the treatment of neoplastic disease (reviewed in Setoguchi et al., J. Investig. Dermatol., 102, 415-421 (1994)). The in vivo approach has been used, among others, for the treatment of cystic fibrosis and neoplastic disease (Setoguchi et al., supra). For the majority of these applications, the coding sequence of the therapeutic gene to be expressed has been placed under the control of an alternative promoter (in particular, a constitutive or inducible promoter), generating a recombinant therapeutic gene.
The predominant approach to gene therapy has employed the retrovirus as a vehicle for gene transfer. However, retroviruses have a number of drawbacks which severely limit their application, particularly in vivo (Mastrangeli et al., J. Clin. Invest., 91, 225-34 (1993); (Burns et al., Proc. Natl. Acad. Sci., 90, 8033-37 (1993)). Consequently, many researchers have turned to the adenovirus as a vector for gene therapy (Horwitz, In: Virology, 2nd Ed., Fields et al., eds., (NY: Raven Press, 1990) 1679-1721; Berkner, K. L., BioTechniques, 6, 606-629 (1988); Ginsberg (ed.) The Adenoviruses (NY: Plenum Press, 1984); Horwitz, supra; Rosenfeld et al., Science, 252, 431-434 (1991); Rosenfeld et al., Cell, 68, 143-155 (1992); Quantin et al., Proc. Natl. Acad. Sci., 89, 2581-2584 (1992); Crystal et al., Nucleic Acids Res., 21, 1607-12 (1993)). Replication-deficient, recombinant adenovirus vectors are highly efficient at transferring genes in vitro and in vivo, and currently are used in a wide variety of applications (see, e.g., Rosenfeld et al. (1991), supra; Rosenfeld et al. (1992), supra; Crystal et al., Nat. Genet., 8, 42-51 (1994); Lemarchand et al., Circ. Res., 72, 1132-1138 (1993); Guzman et al., Circ. Res., 73, 1202-1207 (1993); Bajocchi et al., Nat. Genet., 3, 229-234 (1993); Mastrangeli et al., supra).
Adenoviruses exist as non-enveloped double-stranded DNA viruses (Horwitz, supra). The adenovirus provides an efficient means for transferring biological materials to target cells (Otero et al., Virology, 160, 75-80 (1987); FitzGerald et al., Cell, 32, 607-617 (1983); Seth et al., Mol. Cell Biol., 4, 1528-1533 (1984); Yoshimura, Cell Struct. Funct., 10, 391-404 (1985); Defer et al., J. Virol., 64, 3661-3673 (1990); Rosenfeld et al. (1991), supra; Curiel et al., Proc. Natl. Acad. Sci., 88, 8850-8854 (1991); Rosenfeld et al. (1992), supra; Quantin et al., supra; Curiel et al., Hum. Gene Therapy, 3, 147-154 (1992)). The adenovirus enters cells by a receptormediated endocytosis pathway. In the initial virusreceptor interaction, the adenovirus binds specific receptors present on the cell surface via fibers on its outer surface (Ginsberg, supra; Horwitz, supra; Seth et al., In: Virus Attachment and Entry into Cells, Colwell et al., eds., (DC: American Society for Microbiology, 1986) 191-195). Following attachment, the receptors with bound adenovirus cluster in coated pits, and the virus is internalized within a clathrin-coated vesicle and, subsequently, into an endosomal vesicle, termed an endosome, or receptosome (FitzGerald et al., supra). The adenovirus ultimately is translocated to the nucleus, where it directs the synthesis of nascent nucleic acids (FitzGerald et al., supra; Seth et al. (1984), supra; Seth et al. (1986) supra; Seth et al., J. Virol., 51, 650-655 (1984a); Seth et al., J. Biol. Chem., 259, 14350-14353 (1984b); Seth et al., J. Biol. Chem., 260, 9598-9602 (1985); Seth et al., J. Biol. Chem., 260, 14431-14434 (1985); Blumenthal et al., Biochemistry, 25, 2231-2237 (1986); Seth et al., J. Virol., 61, 883-888 (1987)).
The ability of the adenovirus to easily enter cells has been seized upon as a means of transporting macromolecules into cells (Otero et al., supra; FitzGerald et al., supra; Seth et al. (1984), supra; Yoshimura, supra; Defer et al., supra; Rosenfeld et al. (1991), supra; Curiel et al. (1991), supra; Rosenfeld et al. (1992), supra; Quantin et al., supra; Curiel et al. (1992), supra). There are two means by which such transfer has been effected. First, the adenovirus has been employed to transfer non-viral macromolecules packaged within the adenovirus either in place of, or in addition to, normal adenoviral components (Rosenfeld et al. (1991), supra; Rosenfeld et al. (1992), supra; Quantin et al., supra; Berkner, supra). Second, the adenovirus has been employed to mediate the transfer of non-viral macromolecules either linked to the surface of the adenovirus (e.g., by means of conjugation of the nucleic acid through a polylysine residue to an antibody to adenoviral capsid protein (Curiel et al. (1992), supra)) or in a "bystander" process where the macromolecule is cointernalized and taken along as cargo in the adenoviral receptor-endosome complex (Otero et al., supra; FitzGerald et al., supra; Seth et al. (1984), supra; Yoshimura, supra; Otero et al., supra; Defer et al., supra). Such a bystander process has been employed to enhance the transfer of a variety of non-viral macromolecules including plasmid DNA linked to ligands (Curiel et al. (1991), supra; Curiel et al. (1992), supra; Cotten et al., Proc. Natl. Acad. Sci., 89, 6094-098 (1992)); Rosenfeld et al. (1992), supra; Quantin et al., supra; Cotten et al., J. Viroloqy, 67, 3777-3785 (1993); Wagner et al., Proc. Natl. Acad. Sci., 78, 144-145 (1981)), and plasmid DNA unmodified by nonspecific linkers or by linker-ligand complexes (Yoshimura et al., J. Biolog. Chem., 268, 2300-303 (1993); PCT Application WO 95/21259 (Seth et al.)).
Recently, Setoguchi et al. (Setoguchi et al., supra) disclose adenoviral-mediated gene transfer to adipocytes in vivo of a replication-deficient recombinant adenoviral vector carrying the coding sequence of the .beta.-galactosidase reporter gene under the control of the Rous sarcoma virus long terminal repeat as a promoter. Similarly, Clayman et al. (Clayman et al., Cancer Gene Therapy, 2, 105-111 (1995)) disclose that submucosal injection in mice of a recombinant adenoviral vector carrying a .beta.-galactosidase reporter gene produces scattered staining of adipocytes along the needle track.
Other investigators working with vectors and means of delivery other than adenovirus have transferred genes other than reporter genes to adipocytes in vivo. Specifically, Ross et al. (Ross et al., Genes Devel., 1318-1324 (1993)) disclose the reduction of adiposity via gene transfer to adipose tissue of an attenuated diphtheria toxin A chain under the control of the adipocyte-specific adipocyte P2 (aP2) promoter. Yamaizumi et al. (Yamaizumi et al., Cell, 15, 245-50 (1978)) disclose cell killing through the introduction of diphtheria toxin fragment A, and Gregory et al. (Gregory et al., PCT Application WO 95/11984) disclose means of inducing cell death, such as with use of the conditional suicide gene thymidine kinase. Similarly, Graves et al. (Graves et al., Genes & Development, 5, 428-37 (1991)) and Ross et al. (Ross et al., Proc. Natl. Acad. Sci., 89, 7561-65 (1992); Ross et al., Proc. Natl. Acad. Sci., 87, 9590-94 (1990)) each disclose an adipocyte-specific enhancer located in the 5'-regulatory region of the aP2 gene.
Other references similarly disclose methods for deleting specific cell lineages by cell-specific expression of a toxin gene (Palmiter et al., Cell, 50, 435-43 (1987); Bernstein et al., Mol. Biol. Med., 6, 523-30 (1989); Behringer et al., Genes & Development, 2, 453-61 (1988); Hughes et al., PCT Application WO 92/09616)). The method employed typically calls for microinjecting into fertilized eggs a chimeric gene in which a cell-specific enhancer/promoter is used to drive the expression of a toxic gene product. In a modification of this approach, Hughes et al. (Hughes et al., supra) disclose reduction in the amount of fatty tissues of a host due to introduction of a vector encoding the chicken c-ski protein, which induces myogenic differentiation.
References not involving adenovirus as a means of gene transfer suggest further ways in which adipocytes can be modified in vivo to achieve specific therapeutic aims. Specifically, Spiegelman et al. (Spiegelman et al., J. Biol. Chem., 268(10), 6823-26 (1993)) review the regulation of adipocyte gene expression and suggest "influencing metabolism by controlling adipogenic gene expression" and "interfering! with adipogenesis and systemic metabolism by targeting these key regulators" associated with cell differentiation or obesity. Graves et al. (Graves et al., supra) suggest "the relationship between obesity and diabetes in several obese/diabetic mouse models . . . could be probed by directly suppressing adipose cell formation and/or lipid accumulation through the delivery of toxins or various receptors affecting lipid accumulation". Ross et al. (Ross et al. (1990), supra) disclose the production of transgenic mice containing the adipocyte-specific aP2 gene regulatory region linked to the coding sequence of a reporter gene as a means of monitoring tissue-specific expression and suggest "adipose-directed expression of exogenous genes may be an effective method to alter fat storage and thus directly manipulate the fatness of transgenic animals". Ross et al. (Ross et al. (1992), supra) further disclose the production of transgenic mice containing the adipocyte-specific aP2 gene regulatory region linked to the simian virus 40 (SV40) transforming genes as a means of directing expression of linked exogenous genes, such as oncogenes, to adipose tissue.
Other references also are relevant to adipocyte modification. Specifically, U.S. Pat. No. 5,268,295 (Serrero) relates to a mammalian adipocyte-specific polypeptide, termed p154, which is expressed in high quantities in adipogenic cell lines after differentiation. The '295 patent discloses the murine and human p154 polypeptide, as well as the DNA and RNA molecules coding therefor, methods for its preparation, and antibodies specific for the polypeptide. Flier et al. (Flier et al., Science, 237, 405-8 (1987)) disclose that expression of an adipsin gene and, correspondingly, circulating levels of the serine protease homolog are decreased in obesity. More recently, researchers have demonstrated that the protein product (Ob) of the mouse obese gene causes weight loss, and maintenance of the weight loss, when injected into animals (e.g., reviewed in Barinaga, Science, 269, 475-76 (1995)).
Accordingly, there is a need for an improved means of modifying adipocytes by transferring genes in vivo. It is an object of the present invention to provide such means, as well as vectors for effectuating such means. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.